Age is one of the greatest risk factors of prostate cancer (PCa). DNA-strand breaks and adducts invariably accumulate with advancing age because of constant production of oxygen free radicals during normal metabolic processes and/or continuous assault by environmental hazards such as radical chemicals. Paradoxically, DNA damage-based therapy such as ionizing radiation is a standard treatment for localized PCa and yet treatment often fails and tumors eventually become drug/radiation resistant. Thus, functional genotoxic response pathways are not only important for the maintenance of genomic stability in the normal prostate, but are also critical for the effective treatment of PCa. However, molecular details as to how damaged cells escape to survive genotoxic stress, especially in the absence of a functional p53, are far from elucidated. We and others demonstrated previously that activation of the forkhead transcription factor FOXO1 induces apoptosis in PCa cells, suggesting that FOXO1 is a tumor suppressor protein. This notion is further supported by recent findings from our laboratory, as well as our colleagues', that FOXO1 is a key component of DNA damage response pathways and plays a pivotal role in DNA damage-induced cell cycle arrest and apoptosis. We also demonstrate that CDK1 and CDK2, two key kinases involved in both cell cycle regulation and response to genotoxic stress, interact with and phosphorylate FOXO1 at serine 249 (S249), thereby resulting in FOXO1 inactivation. We demonstrate in this proposal that CDK-mediated phosphorylation of FOXO1 is abrogated in PCa cells treated with high but not low doses of DNA-damaging agents, suggesting that CDK phosphorylation of FOXO1 may contribute to the resistance of prostate cancers to DNA-targeted therapy. We further demonstrate that replacement of endogenous FOXO1 with a CDK phosphorylation-resistant mutant of FOXO1 sensitizes PCa cells to death induced by a clinically relevant dose of radiation. Given that the activities/levels of CDK1 and CDK2 are often elevated in human prostate cancers, these findings suggest that a functional FOXO1 is critical for the genotoxic stress response in the prostate and that inhibition of FOXO1 due to increased activity of CDK1 and/or CDK2 may contribute to prostate tumorigenesis and therapy resistance by promoting cancer cell survival. The unifying hypothesis of this study is that CDK1 and CDK2 enhance PCa cell survival and resistance to DNA-targeted therapies via phosphorylation-mediated inhibition of the pro-apoptotic function of FOXO1. To test this hypothesis, we propose to dissect the molecular mechanism of how phosphorylation of FOXO1 by CDK1 and CDK2 leads to the loss of function of FOXO1 (Aim 1). Moreover, the importance of phosphorylation-mediated inactivation of FOXO1 due to aberrant activation of CDK1 and CDK2 for PCa growth, survival and resistance to therapy will be assessed using orthotopic xenograft models (Aim 2). Finally, a selective CDK1/2 phosphorylation-antagonistic motif of FOXO1 will be defined and the role of this motif in regulation of the pro-apoptotic function of FOXO1 as well as the growth and survival of PCa cells will be evaluated (Aim 3). Studies in this proposal will elucidate the precise role of CDK1/2 phosphorylation- mediated inhibition of FOXO1 in PCa survival and resistance to DNA-targeted therapy, and further provide insights into the function of the newly defined genotoxic-stress-response mechanism in ag associated prostate carcinogenesis and PCa prevention and therapy. PUBLIC HEALTH RELEVANCE: Age is one of the greatest risk factors of prostate cancer, and DNA-strand breaks are implicated in aging-associated prostate tumorigenesis as well as prostate cancer therapy. This project is designed to elucidate the role of inhibition of the FOXO1 tumor suppressor protein by the key cell cycle driving kinases CDK1 and CDK2 in prostate cancer growth, survival and resistance to DNA-targeted therapies. Findings from this study will not only enhance our understanding of age-related prostate cancinogenesis, but could also provide new molecular targets to devise strategies for prostate cancer prevention and therapy.